ABSTRACT: In mammalian cells, alternatively splicing (AS) of RNA transcripts allows extensive diversification and tailoring of cells' protein repertoires, yet this process frequently goes awry in diseases ranging from cancer to neurological disorders. Transcription rate strongly regulates AS, presumably through changing the relative ?windows of opportunity? for competing splicing reactions. Decades of work have elucidated splicing chemistry, spliceosomal assembly and many AS mechanisms, yet how transcription elongation interacts with splicing machinery remains largely unknown. Constitutive and alternative splicing require the C-terminal domain (CTD) of RNA polymerase II (Pol II), consisting of heptapeptide repeats (YSPTSPS). The central hypothesis of this proposal is that transcriptional pausing events and specific Pol II CTD phosphisoforms control splicing in yeast and regulate alternative splicing in human cells. Preliminary data obtained with a nucleotide-resolution approach that maps Pol II density genome-wide, native elongating transcript sequencing (NET-seq), reveals distinct Pol II pausing signatures in retained and skipped exons, indicating that Pol II recognizes exons with different processing fates. In yeast and in humans, NET-seq detects strong pausing signatures at exon-intron junctions, which may mediate connections between elongation and splicing. Moreover, a quantitative mass spectrometry analysis of Pol II complexes demonstrates a role for CTD phosphoisoforms in coordinating transcription and splicing. In Aim 1, fundamental coupling mechanisms will be explored in yeast, where splicing is solely constitutive and powerful yeast genetics will be exploited. Specifically, how Pol II CTD phosphorylation connects transcription to splicing will be investigated. High-throughput genetics and proteomics approaches will identify the factors that associate with Pol II CTD phosphoisoforms and connect transcription to splicing. These factors will be characterized using high-resolution genomics approaches, NET-seq and RNA-seq. Many splicing mechanisms are conserved from yeast to human, and the proposed yeast studies will inform efforts in Aims 2 and 3 to determine more complex transcription-splicing connections in human cells. In Aim 2, how control of transcriptional pausing is linked to alternative splicing in human cells will be established. NET-seq and RNA-seq analyses of genetic perturbations of nine factors reported to influence both transcription and splicing will determine how alterations in transcriptional pausing correlate with AS outcomes. In Aim 3, the human Pol II CTD phosphoisoforms that connect transcription to splicing will be determined. Tandem mass-tag mass spectrometry of different human Pol II CTD phosphoisoforms will identify modifications and factors that connect transcription and splicing. Genetic perturbation experiments followed by NET-seq and RNA-seq analysis will determine how these factors influence transcription and alternative splicing. The completion of these Aims will have the positive impact of determining the core factors responsible for couple transcription and splicing, a critical first step towards understanding how transcription regulates AS.